Staphylococcus aureus is a major human pathogen that causes significant morbidity and mortality in both hospital- and community-acquired infections. The appearance of multidrug-resistant strains has compounded this problem, galvanizing efforts aimed at identifying novel therapeutic targets. One promising area for the development of novel antimicrobials is S. aureus heme metabolism, as this process is vital to staphylococcal pathogenesis. S. aureus acquires heme from hemoglobin to satisfy its nutrient iron requirement. Once heme is internalized, it is enzymatically degraded, resulting in the production of small molecule chromophores with as- yet-undetermined functions in bacteria. Intriguingly, most bacterial pathogens that utilize heme as a nutrient iron source can also synthesize heme through the activity of a dedicated synthesis pathway. Heme synthesis is conserved in virtually all cells, yet the process by which heme synthesis is regulated is largely unknown. We hypothesize that heme catabolites have a critical role in staphylococcal physiology. We also predict that S. aureus heme synthesis is regulated to adjust to the amount of heme and heme catabolites within the cell. Notably, we have discovered a small molecule activator of an enzyme within the heme synthesis pathway and this molecule sensitizes S. aureus to light, establishing a valuable probe to study heme synthesis as well as a candidate molecule for antibiotic development. In this grant application, we propose experiments to test this model. In Aim 1 we will define the function of heme degradation products in S. aureus. In Aim 2 we will define the contribution of regulated heme synthesis to staphylococcal pathogenesis. Finally, Aim 3 studies will determine the utility of small molecule-dependent activation of heme synthesis as a molecular tool as well as a candidate therapeutic strategy. Together, these proposed experiments will provide a mechanistic understanding of the coordination of heme synthesis and degradation in S. aureus, and exploit this knowledge to generate candidate antimicrobials with efficacy against numerous Gram positive pathogens.